Differential gene expression, single nucleotide polymorphisms (SNPs), mutations and genetic information associated with diseases including from pathogenic bacteria and viruses have been elucidated. Those differences or variations in genetic information provide differences between individuals, and determine the development of genetic diseases and susceptibility to diseases. Commonly used techniques for analyzing variation or gene expression include DNA sequencing, RFLP (restriction fragment length polymorphism), allele specific polymerase chain reaction (PCR), southern blot, northern blot, and the like [Present and future of rapid and/or highthroughput methods for nucleic acid testing, Gyorgy csako, 2005, Clinica Chimica Acta 1-25]. However, those techniques are time- and cost-consumptive, and labor- and skill-intensive, only one gene or variation can be analyzed at one time, and gel electrophoresis, which is cumbersome, should be involved.
As a novel analysis system which can overcome the drawbacks of the prior genetic analysis methods, DNA chip or DNA microarray technique has been developed [Single nucleotide polymorphism discrimination assisted by improved base stacking hybridization using oligonucleotide microarrays, Wang D. et al., 2003, Biotechniques, 35(2), 300-306]. DNA chip has DNA probes designed on the basis of known genetic information immobilized on a solid surface. Typically the hybridization with a target nucleic acid to be analyzed on the chip is detected with fluorescence. By using the DNA chip, a variety of genetic information can be analyzed by a single experiment, so that it is extremely useful for diagnosis of diseases [Development and evaluation of a highly sensitive human papillomavirus genotyping DNA chip, Kim K et al., 2006, Gynecologic Oncology 100, 38-43]. DNA chips have been known as the most efficient analysis and diagnosis system among those having been developed hitherto, but still involve technical problems as follows:
First, stability of a DNA chip product is low due to low biological (such as against nuclease, etc.) and chemical (such as against acid, base, etc.) stability of DNA probes.
Second, single nucleotide differences such as SNP, point mutation, etc. can hardly be discriminated with accuracy.
Third, the length of target nucleic acid is limited for hybridization with oligonucleotide probes on a chip.
In using probes immobilized on a support, as in a DNA chip, access to the probes becomes more difficult and so efficiency of hybridization is reduced, as the size of the target nucleic acid is increased. Thus, target nucleic acid, which is not too much long, should be applied to the hybridization. If the length of the target nucleic acid is about 200 base pairs (bp) or longer, the efficiency of hybridization abruptly decreases to reduce perfect match signal, and thus, discrimination from background signal is not easy. The target nucleic acid with the length of longer than 400 bp generates almost no perfect match signal, and so cannot be analyzed [Optimization of fragmentation conditions for microarray analysis of viral RNA, Martin et al., 2005, Analytical biochemistry, 347, 316-323; and Correlation between microarray DNA hybridization efficiency and the position of short capture probe on the target nucleic acid, Regis et al., 2005, BioTechniques, 39, 89-96]. In order to overcome the problems, amplification as separated short fragments when the targets are scattered, long-size amplification followed by fragmentation with restriction enzyme, and amplification of genome followed by small-size amplification with individual specific primers, or the like has been employed [Toward genome-wide SNP genotyping, Ann-Christine Syvanen, 2005, Nature genetics, 37, S5-S10; and Assessing Genetic Variation: Genotyping Single Nucleotide Polymorphism, Ann-Christine Syvanen, Nature, 2001, 2, 930-942]. However, those are cumbersome and inefficient, requiring much time and effort and high cost for manufacturing the target nucleic acid into small fragments which are capable of hybridization. Further, non-specific signal may be increased from reaction with unreacted residual target nucleic acids.
Various DNA analogues have been developed to overcome instability of DNA itself. Among them, PNA (peptide nucleic acids) was developed by Neilson in 1991 [Peptide nucleic acid, PNA, sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide, P. E. Neilson et al., 1991, Science, 254, 1497-1500]. As shown in FIG. 1, phosphodiester bond of DNA has been replaced by peptide bond in PNA. PNA comprises adenine, thymine, guanine and cytosine as DNA, so that PNA can base specifically hybridize with DNA or RNA. In particular, differently from natural nucleic acids which electrically repel one another due to phosphate backbone having negative charge, PNA has peptide backbone having no charge, and thus, as compared with DNA, it forms stronger bond with DNA upon hybridization, and the bond is not influenced by salt concentration. Further, since PNA is not degraded by biological degrading enzymes such as nuclease and protease, it is more stable than DNA or RNA. Thus, PNAs, which can complementarily bind with natural nucleic acids, and has high binding strength and stability upon hybridization, have been utilized in genetic analysis or diagnosis [PNA for rapid microbiology, Stender H et al., 2002, Journal of Microbiological Methods, 48, 1-17], [Peptide nucleic acids on microarrays and other biosensors, Brandt O et al., 2004, Trends in Biotechnology, 22, 617-622; and Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support, Frdric R Raymond et al., 2005, BMC technology, 5, 1-5].
As studies taking advantage of the biological stability of PNA was reported a process for discriminating SNP by means of FRET (fluorescence resonance energy transfer), wherein a cationic polymer is bound with an anion of DNA upon the hybridization of PNA and DNA, while the mismatched region between PNA and DNA, if any, is removed by S1 nuclease, one of nucleases [SNP detection using peptide nucleic acid probes and conjugated polymers: Applications in neurodegenerative disease identification, Brent S et al., 2005, Proceedings of the National Academy of Sciences 102, 34-39]. Further, a process has been reported, wherein one or two PNA probe(s) is (are) hybridized with a target nucleic acid in a microtube, and then, treated with nuclease to remove the target nucleic acids with the base sequence mismatching with PNA probes, and a fluorophore is attached to target nucleic acids completely hybridized with PNA probes, to observe with naked eyes or mass spectrometry [Detection of single nucleotide polymorphisms by the combination of nuclease S1 and PNA. Sheng Ye et al., 2002, Nucleic Acid Research Supplement No. 2, 235-236; and PNA for one base differentiating protection of DNA from nuclease and its use for SNP detection. Makoto Komiyama et al., 2002, Journal of American Chemical Society 2003, 125, 3758-3762].
However, according to the above-described processes, hybridization is performed in a homogeneous solution, and thus, it occurs regardless of the size of the target nucleic acid. According to the processes, nuclease is simply added after hybridization to remove the mismatched region between target nucleic acids and PNA probes, thereby increasing the specificity. In those processes, a target nucleic acid with long length was not used since one or two PNA probes was (were) used at one time to analyze one genetic variation at one time.
Korean Patent Registration No. 436554 (issued on Jun. 8, 2004) disclosed a process for increasing the detection sensitivity of hybridized nucleic acid by applying nuclease to a conventional DNA chip. The process involves removing unhybridized single stranded DNA probes among the immobilized DNA probes, by using nuclease. Specifically, the process involves removing unhybridized DNA probes with the immobilized 5′-terminal on the substrate and the exposed 3′-terminal hydroxyl group, by using exonuclease I which cannot recognize the terminal of a double stranded DNA but can recognize only the 3′-terminal of a single stranded DNA, and hydrolyze only a single stranded DNA with 3′-terminal OH group. Thus, the process is completely different from fragmentation or selective degradation of target nucleic acids by using nuclease. This process may reduce non-specific signal from background signal, but cannot reduce non-specific signal from single nucleotide mismatch. Accordingly, the process can be hardly applied to detect SNP or mutation.